Myosin X is an unconventional myosin that has been implicated in filopodial development in mammals. We have recently characterized its steady-state and transient state MgATPase activity. Myosin X contains a region of predicted coiled-coil 120 residues long. However, the highly charged nature, and pattern of charges in the proximal 36-residues, appears incompatible with coiled-coil formation. We have shown that this domain forms a stable single alpha-helical domain (SAH domain) and functions to extend the neck region of the myosin which forms part of the lever arm of myosin. Thus, the powerstroke is lengthened. We have carried out optical trapping experiments with a forced dimer of myosin X where a leucine zipper was added at the end of the predicted coiled-coil region. This molecule is shown to be dimeric by electron microscopy. We have measured its mechanical properties using optical trapping nanometry and find that it has a power stroke of about 17 nm. Increasing the calcium concentration increased the power stroke size to 23 nm consistent with three IQ motifs and a SAH domain in the lever arm. We believe the increase power stroke length is due to binding of an additional calmodulin to the third IQ motif in the presence of calcium. The attachment lifetimes are consistent with the ADP release rate measured in vitro. At low trap stiffness, the myosin X shows processive movement (forward and backward steps) occurring with steps of about 35 nm. This is consistent with electron micrographs showing the molecule attached by two head to actin monomer that are separated by 36 nm in the actin filament. In collaboration with a former postdoc, Takeshi Sakamoto, we show that single molecule TIRF assays show that the molecule moves processively along actin in the absence of load with 36 nm steps. We examined the movement of myosin X on parallel bundles. The myosin walks predominantly along a single actin filament, but takes frequent side steps onto adjacent filaments

Project Start
Project End
Budget Start
Budget End
Support Year
13
Fiscal Year
2013
Total Cost
$236,277
Indirect Cost
Name
National Heart, Lung, and Blood Institute
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